S-nitrosoprotein preparation and production method therefor

ABSTRACT

A protein is efficiently nitrosylated with nitrogen monoxide by merely mixing S-nitrosoglutathione as a nitrogen monoxide donor with a protein solution containing a stabilizing agent comprising at least one compound or a combination of plural compounds selected from an N-acetylamino acid, a fatty acid, and a fatty acid salt. The method that enables efficient NO addition to a cysteine residue in un-nitrosylated protein without changing the structure of protein and hence provides NO to a living organism.

TECHNICAL FIELD

The present invention relates to an S-nitrosoprotein preparationcontaining a stabilizing agent and to a production method therefor. Morespecifically, the present invention relates to an S-nitrosoproteinpreparation containing nitrogen monoxide which is efficiently producedby merely mixing S-nitrosoglutathione as a nitrogen monoxide donor witha protein solution containing a stabilizing agent.

BACKGROUND ART

Nitrogen monoxide (hereinafter abbreviated as NO) has been widelystudied in various fields such as applications to control physiologicalfunctions, clinical episodes, and therapy. In particular, in ischemicdisorders or at the time of organ transplantation, various disorders arecaused by endogenous reduction in NO production due to substratedepletion accompanying blood flow decrease, so that exogenous NOsupplementation (called NO supplementation therapy) is indispensable.Although among others NO inhalation therapy has been tried as the NOsupplementation therapy, the current situation is that NO disappears inan extremely short time in a living organism and selective obtention ofadvantageous effects is difficult because of NO itself having highreactivity and diversity (Ignarro L. J. et al., Pharm Res., 1989, 6,651-659).

NO is known to react, in a living organism, with a thiol group(hereinafter, abbreviated as an SH group) of a protein or the like andconverted into S-nitrosothiol (hereinafter, abbreviated as RS—NO), whichmaintains a relatively stable state and functions as an NO reservoir,thus participating in control of NO concentration in the living organism(Ignarro L. J. et al., J. Pharmacol. Exp. Ther., 1981, 218 739-749).

Usefulness of nitrosylated proteins has been reported. Analyses on theefficiency of nitrosylation and antibacterial activity of variousbacteria-infected model animals using albumin variants having a mutationin one or more amino acid residues in the constituent amino acidsequences indicated that the albumin variants are efficientlynitrosylated and the nitrosylated products exhibit more potentantibacterial activity than NO and low molecular weight nitrosothiol (JP2005-206577 A, US2005/0222026).

On the other hand, albumin can be used as an NO transport protein. Inspite of having a free cysteine residue at the 34th position, albumin isknown to have only a low reactivity, so that attempts to allow albuminand an NO donor to react to promptly obtain S-nitrosylated albumin fail.Then, to add NO to albumin, a method is known in which the bindingefficiency is increased by adding a suitable chemical modulator toalbumin or the reaction efficiency is increased by mutating a part ofamino acids of albumin to cysteine. These methods involve changing theinherent structure of albumin, which causes the problem that thebehavior of albumin in the living organism may be changed.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a nitrosoproteinpreparation and to provide a production method that enables efficient NOaddition to a cysteine residue in an un-nitrosylated protein withoutchanging the structure of the protein and hence to provide NO to aliving organism.

The inventors of the present invention have made extensive studies witha view to achieving the above-mentioned object and as a result, theyhave found that addition of a stabilizing agent such as a fatty acid,fatty acid salt or N-acetylamino acid to a protein allows reaction of athiol group of the protein with NO to provide S-nitrosoprotein, thusaccomplishing the present invention. In particular, addition of astabilizing agent such as a fatty acid, fatty acid salt or N-acetylaminoacid to albumin permits reactivity of cysteine at the 34th position tobe increased, allowing ready reaction of cysteine in the albumin with NOto provide S-nitrosoalbumin, thus accomplishing the present invention.

That is, the present invention provides:

(1) an S-nitrosoprotein preparation containing a stabilizing agentincluding at least one compound or a combination of compounds selectedfrom an N-acetylamino acid, a fatty acid or a fatty acid salt;

(2) the S-nitrosoprotein preparation according to item (1), in which theN-acetyl amino acid is N-acetyltryptophane or N-acetylmethionine;

(3) the S-nitrosoprotein preparation according to item (1), in which thefatty acid is oleic acid or caprylic acid;

(4) the S-nitrosoprotein according to item (1), in which the fatty acidsalt is sodium caprylic acid;

(5) a method of producing an S-nitrosoprotein preparation comprisingadding a nitrogen monoxide donor to a protein solution containing astabilizing agent including at least one compound or a combination ofcompounds selected from N-acetylamino acid, a fatty acid or a fatty acidsalt;

(6) the production method according to item (5), in which the nitrogenmonoxide donor is S-nitrosoglutathione;

(7) an improving agent for an ischemic disorder containing theS-nitrosoprotein preparation according to any one of items (1) to (4),in which the S-nitrosoprotein is S-nitrosoalbumin;

(8) an organ protecting agent for organ transplantation containing theS-nitrosoprotein preparation according to any one of items (1) to (4),in which the S-nitrosoprotein is S-nitrosoalbumin;

(9) a pharmaceutical composition containing the S-nitrosoproteinpreparation according to any one of items (1) to (4), in which theS-nitrosoprotein is S-nitrosoalbumin; and

(10) a pharmaceutical composition according to item (9), in which thepharmaceutical composition is obtained by mixing an albumin solutioncontaining a stabilizing agent with a nitrogen monoxide donor from 24hours before administration to just before administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating change of SH group sensitivity in an HSAsolution in the presence of a fatty acid versus time.

FIG. 2 shows a graph illustrating change of the NO addition rate versustime of an HSA solution when a nitroso-HSA sample was stored undervarious conditions.

FIG. 3 is a graph illustrating the spectra of a nitroso-HSA sample in afar-ultraviolet region (Far-UV CD spectra).

FIG. 4 is a graph illustrating the spectra of a nitroso-HSA sample in anear-ultraviolet region (Near-UV CD spectra).

FIG. 5 illustrates a band of SDS-PAGE electrophoresis of anS-nitroso-HSA sample after low temperature sterilization treatment.

FIG. 6 shows charts illustrating variation of levels of ALT and AST of anitroso-HSA sample after low temperature sterilization treatment beforeischemia and after reperfusion in rats.

The S-nitrosoprotein preparation of the present invention, whichcontains one or a combination of stabilizing agents selected from thegroup consisting of an N-acetylamino acids, a fatty acid, and a fattyacid salt, can increase the binding efficiency between an SH group inthe protein and nitrogen monoxide, and increase stability. For example,when the protein is albumin, there occurs substantially no structuralchange and a stable S-nitrosothiol conformation can be obtained.

An S-nitrosoalbumin preparation serves as a donor of NO for variousdisorders caused by endogenous reduction in NO production due tosubstrate depletion accompanying a blood flow decrease in ischemicdisorders or during organ transplantation, and protects ischemic siteorgan derived from activated oxygen species. Further, S-nitrosoproteinpreparations can be expected to exhibit biological activities such asimprovement of blood flow in liver tissue, suppression of neutrophilinfiltration, neutrophil apoptosis activity, etc. Moreover, a protein,for example, albumin, undergoes substantially no structural change andhence is not recognized as a heterologous protein, so that the protein,for example, albumin, can be used safely in a human body.

In the present invention, nitrosylation means addition of a nitrosogroup (—NO) to a protein. Nitrosylation of the protein is preferablyaddition of a nitroso group to a thiol group in the protein, which canbe achieved by a known method which involves reaction with a nitrogenmonoxide donor such as nitrous acid salts, S-nitrosoglutathione(hereinafter, abbreviated as GSNO), S-nitrosocysteine,S-nitroso-N-acetyl-DL-penicillamine, or propylamine NONOate(diazoniumdiolate).

In the present invention, the protein to be nitrosylated is notparticularly limited and commonly used proteins can be used. Forexample, any protein having a thiol group such as albumin, globulin,heparin, hemoglobin, or a1-antitrypsin may be used. Among these,albumin, globulin, and heparin that are present in blood are preferable.Proteins which are produced by genetic engineering may also besatisfactorily used. When used as a drug, those proteins derived fromhumans are preferable and in this case, the proteins may be either onescollected from individuals or ones produced by genetic engineering.Human-derived albumins are preferable irrespective of whether they arecollected from individuals or produced by genetic engineering.

The stabilizing agent to be added to the protein of the presentinvention can be at least one compound or a combination of compoundsselected from an N-acetylamino acid, a fatty acid and a fatty acid salt.Presence of the stabilizing agent enables nitrosylation in whichnitrogen monoxide is added to, for example, the thiol group at the 34thposition of the amino acid sequence of albumin and conversion of theprotein into a protein that is stable after the nitrosylation.

The fatty acid or fatty acid salt to be added to the protein is notparticularly limited and among those represented by the general formulaC_(n)H_(2n+1)COOH, those represented by the general formula in which n=2to 30 are preferable, more preferably, those represented by the generalformula in which n=4 to 20, and more preferably, those represented bythe general formula in which n=6 to 10, are suitable. Examples thereofinclude caprylic acid, caproic acid, and capric acid. Among these,caprylic acid is preferable. Fatty acid salts include sodium salts,potassium salts, etc. thereof.

The fatty acid(s) or fatty acid salt(s) or N-acetylamino acid(s) orcombination thereof can be added to the protein in a concentration of0.01 to 100 mM, preferably 0.1 to 50 mM, and more preferably 1 to 20 mM.The method of adding the fatty acid(s) or fatty acid salt(s) orN-acetylamino acid(s) or combination thereof to the protein is notparticularly limited. The fatty acid(s) or fatty acid salt(s) orN-acetylamino acid(s) or combination thereof may be contained in theprotein in advance or suitable amounts of the fatty acid(s) or fattyacid salt(s) or N-acetylamino acid(s) or combination thereof may beadded to the protein. When the fatty acid(s) or fatty acid salt(s) orN-acetylamino acid(s) or combination thereof is (are) in a mixed statewith the protein, a sufficiently good environment is provided for thenitrosylation of proteins.

N-acetylamino acid(s) contained as a stabilizing agent includes aminoacid components such as N-acetyltryptophane and N-acethylmethionine.

Pharmaceutically acceptable excipients cause no problems in thenitrosylation and can be added to the S-nitrosoprotein preparation ofthe present invention.

In the nitrosylation, a thiol group of the protein is nitrosylated byadding a nitrogen monoxide donor for nitrosylation to a solutioncontaining a stabilizing agent, for example, a caprylic acid, and aprotein such as albumin.

The nitrogen monoxide donor includes a nitrous acid salt, aS-nitrosoglutathione (GSNO), an S-nitrosocysteine, anS-nitroso-N-acetyl-DL-penicillamine, and a propylamine NONOate. The GSNOis preferable.

When a protein is nitrosylated, the content of the nitrogen monoxidedonor can be 0.01 to 50 mM, preferably 0.1 to 30 mM, and more preferably1 to 10 mM.

The nitrosylation of a protein can be performed at a temperature of 4 to40° C., preferably 15 to 40° C., and more preferably 25 to 37° C. for atime of 1 to 360 minutes, preferably 10 to 300 minutes, and morepreferably 20 to 180 minutes.

Among proteins, a chemically non-modified albumin is nitrosylated at thethiol group at the 34th position of the amino acid sequence of thechemically non-modified albumin. In this case, the nitrosylation isperformed under very mild conditions without an accompanyingintermolecular cleavage of amino acids or an intramolecular cleavage ofamino acids and, therefore, it is seldom the case that an originalfunction or a steric structure of albumin is damaged.

For example, albumin has 17 sites where the thiol groups undergointramolecular crosslinking and the nitrosylation can be performed inthe presence of the stabilizing agent after the internal crosslinking isreductively cleaved.

When the S-nitrosoprotein of the present invention is S-nitrosoalbumin,S-nitrosoalbumin itself is effective to an ischemic reperfusion injurymodel and, therefore, can be used as an improving agent for ischemicdiseases.

When the S-nitrosoprotein of the present invention is S-nitrosoalbumin,S-nitrosoalbumin itself has an anti-apoptosis effect or an inductionactivity for antioxidation and, therefore, can be used as an organprotecting agent at the time of organ transplantation.

The methods of administering the improving agent and the protectingagent of the present invention include oral administration, subcutaneousinjection administration, and intravenous administration. However, thepresent invention should not be considered to be limited thereto.

The S-nitrosoprotein preparation of the present invention can be anS-nitrosoalbumin preparation and a pharmaceutical composition containingthe S-nitrosoalbumin preparation can be formulated. Examples of thepharmaceutical composition include a composition that contains commonlyused components constituting tablets such as an excipient, a lubricant,a binder and the S-nitrosoalbumin preparation of the present invention;a composition that contains commonly used components constituting aninjectable agent, such as a solvent, a tonicity agent, a pH adjuster,and an antiseptic agent and the S-nitrosoalbumin preparation of thepresent invention.

Further, there may be exemplified a pharmaceutical composition thatincludes an albumin solution containing a stabilizing agent and anitrogen monoxide donor which are separately packaged and mixed andadjusted during a period from 24 hours before administration to justbefore administration. That is, S-nitrosoalbumin is more stabilized thanconventional one by at least one compound or a combination of pluralcompounds selected from N-acetylamino acids, fatty acids, and fatty acidsalts, and S-nitrosoalbumin preparation can be supplied in a more stablestate by formulating S-nitrosoalbumin as a pharmaceutical compositionthat can be mixed with a nitrogen monoxide donor and adjusted during aperiod from 24 hours before administration to just beforeadministration. When albumin is nitrosylated without the stabilizingagent, the albumin is not nitrosylated (see, Comp. Ex. 2).

EXAMPLE Reference Example 1

A 3 mM solution of a recombinant human serum albumin (hereinafter,abbreviated as HSA) solution containing 16.0 mM caprylic acid and 18.6mM N-acetyltryptophane was measured for absorbance at a wavelength of405 nm using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (hereinafter,abbreviated as DTNB method) to confirm the sensitivity of SH group ofHSA. The result obtained is illustrated in FIG. 1.

In FIG. 1 the ordinate axis indicates absorbance at 405 nm of3-carboxy-4-nitrosothiophenolate ions liberated by the reaction of DTNBwith an SH group.

As illustrated by the time line indicated by the symbol ▪ in FIG. 1, inthe case of the HSA solution, the reaction reached a plateau in about 30minutes, which confirmed that the SH group of albumin in the HSAsolution containing 16.0 mM caprylic acid had a high sensitivity.

Comparative Reference Example 1

To the HSA solution containing 16.0 mM caprylic acid and 18.6 mMN-acetyltryptophane activated carbon was added to obtain defattedalbumin of the same concentration, which then was confirmed forsensitivity of the SH group in the same manner as that in Example 1. Theresult obtained is illustrated in FIG. 1.

As illustrated by the time line indicated by the symbol ●, in the caseof defatted albumin, the absorbance increased moderately and did notreach a plateau even after 30 minutes.

Example 1

A 300 μM HSA solution containing 1.6 mM caprylic acid and 1.86 mMN-acetyltryptophane was prepared. To the HSA solution was added GSNO inan amount five times that of the HSA by mole ratio and the resultant wasallowed to react at 37° C. for 1 minute to obtain an S-nitroso-HSAsample. Then, aliquots of sample were subjected to storage tests for 10days under conditions of 4° C. with light shielding, 4° C. without lightshielding, 25° C. with light shielding, and 25° C. without lightshielding, respectively, and NO concentrations were measured with timeby an HPLC-flow reaction system method using a Griess reagent to confirmthe nitrosylated state of HSA.

The result obtained is illustrated in FIG. 2. The ordinate axisindicates S-nitroso moiety, that is, NO addition rate. The symbol---∘--- indicates a storage condition of 4° C. with light shielding; thesymbol --●-- indicates a storage condition of 4° C. without lightshielding; the symbol ---□--- indicates a storage condition of 25° C.with light shielding; and the symbol --▪-- indicates a storage conditionof 25° C. without light shielding. The ordinate axis indicates NOconcentration per mol HSA.

While under the conditions of 4° C. with light shielding and 4° C.without light shielding, no great difference was observed in residualratio of nitroso group, whereas disappearance of NO when stored at 25°C. was extremely rapid under the condition without light shielding ascompared with the condition with light shielding. In this study, noremoval operation for GSNO, that is, the NO donor, was performed, whichwould lead to a gradual increase in residual ratio until day 5.

Comparative Example 1

All the procedures of Example 1 were repeated except that a 300 μM HSAsolution containing neither 1.6 mM caprylic acid nor 1.86 mMN-acetyltryptophane was used. As a result, without the need of checkingstability, nitrosylation itself of HSA was not observed.

Example 2

A 300 μM HSA solution containing 1.6 mM caprylic acid and 1.86 mMN-acetyltryptophane was prepared. To this was added a 1.5 mMS-nitrosoglutathione (GSNO) solution and the resultant was mixed at 37°C. for about 30 minutes with light shielding to obtain an S-nitroso-HSAsample. Then, the sample was measured for NO concentration by anHPLC-flow reactor system method using a Griess reagent to confirm thenitrosylation of HSA.

Experimental Example 1

To confirm the structure of S-nitrosoalbumin obtained in Example 2,circular dichroic spectrum (hereinafter abbreviated as CD-spectrum) wasmeasured (using a Jasco corporation J-720 Model spectropolarimeter). Theresults obtained are illustrated in FIG. 3 and FIG. 4.

In FIG. 3, the ordinate axis indicates [θ], that is, ellipticity permole and the abscissa axis indicates wavelength. HSA indicated by abroken line indicates non-nitrosylated albumin and S—NO—HSA indicated bya solid line indicates nitroso-HSA sample.

In FIG. 4, the ordinate axis indicates [θ], that is, ellipticity permole and the horizontal axis indicates wavelength. The abscissa axisindicates wavelength. HSA indicated by a broken line indicatesnon-nitrosylated albumin and S—NO—HSA indicated by a solid lineindicates nitroso-HSA sample.

FIG. 3 indicates that the nitroso-HSA sample in a far-ultraviolet regionthat reflects a secondary structure exhibited substantially the sameCD-spectrum as that of HSA, thus confirming that the sample maintainedthe inherent a-helix structure of albumin.

In addition, FIG. 4 indicates that the nitrosoalbumin sample in anear-ultraviolet region, which reflects a tertiary structure, exhibitedsubstantially the same CD-spectrum as that of HSA. As mentioned-above,the nitroso-HSA sample obtained in Example 3 maintained the originalstructure of HSA.

Experimental Example 2

The S-nitroso-HSA sample obtained in Example 2 was subjected to lowtemperature sterilization at 60° C. for 10 hours. The S-nitroso-HSAsample after the low temperature sterilization treatment was confirmedas to whether or not the protein was aggregated in the presence orabsence of dithiothreitol (hereinafter, abbreviated as DTT) by SDSpolyacrylamide gel electrophoresis (hereinafter, abbreviated asSDS-PAGE). The result obtained is illustrated in FIG. 5.

In FIG. 5, “1: Molecular weight marker” indicates a trypsin inhibitor(molecular weight: 20,100), carbonic anhydrase (molecular weight:30,000), ovalbumin (molecular weight: 45,000), albumin (molecularweight: 66,000), and phosphorylase b (molecular weight: 97,000) asmolecular weight markers. “2: HSA” indicates a non-nitrosylated albumin.“3: S—NO—HSA” indicates an S-nitroso-HSA sample. “4: S—NO—HSA (60° C.,10 h)” indicates a nitroso-HSA sample after a low temperaturesterilization treatment.

The nitroso-HSA sample after the low temperature sterilization treatmentdid not show aggregation like HSA and nitroso-HSA (hereinafter,abbreviated as S—NO—HSA). That is, the nitroso-HSA sample was stableeven against low temperature sterilization.

Experimental Example 3

Nitroso-HSA sample obtained in Example 2 was evaluated using an ischemiareperfusion injury model obtained by median incision of the abdomen of arat under etherization to expose the liver, simultaneously clipping theportal vein and hepatic artery so that only the middle lobe and leftlobe were ischemic to block the blood flow for 45 minutes and thenrecovering the blood flow. As the evaluation, after 60 minutes, ASTinduced and plasma level of AST were measured.

That is, after the reperfusion, 0.1 mmol/kg or 0.5 mmol/kg of thenitroso-HSA sample obtained in Example 2 was separately and immediatelyintravenously administered. For comparison, 0.5 mmol/kg HSA wasseparately administered in the same manner as above. The result obtainedis illustrated in FIG. 6.

In the ordinate axes in FIG. 6, ALT indicates the amount ofalanine-amino group transferase while AST indicates the amount ofaspartic acid-amino group transferase. HSA 0.5 mmol/kg expressed by awhite bar indicates administration with a 0.5 mmol/kg non-nitrosylatedalbumin; S—NO—HSA 0.1 mmol/kg expressed by a black bar indicatesadministration with a 0.1 mmol/kg nitroso-HSA sample; and S—NO—HSA 0.5mmol/kg expressed by a bar with dots indicates administration with a 0.5mmol/kg nitroso-HSA sample.

FIG. 6 indicates that in the group administered with the 0.5 mmol/kgnitroso-HSA sample, increases in alanine-amino group transferase value(hereinafter, abbreviated as ALT value) and asparagine-amino grouptransferase value (hereinafter, abbreviated as AST value), respectively,were significantly suppressed as compared with the group administeredwith HSA. This indicates that the nitroso-HSA sample had an effect onthe liver ischemia reperfusion injury.

This application claims priority of Japanese patent application No.2006-227427 filed Aug. 24, 2006, which is incorporated herein byreference.

1. An S-nitrosoprotein preparation containing a stabilizing agentcomprising at least one compound or a combination of compounds selectedfrom an N-acetylamino acid, a fatty acid, and a fatty acid salt.
 2. TheS-nitrosoprotein preparation according to claim 1, wherein the N-acetylamino acid is N-acetyltryptophane or N-acetylmethionine.
 3. TheS-nitrosoprotein preparation according to claim 1, wherein the fattyacid is oleic acid or caprylic acid.
 4. The S-nitrosoprotein preparationaccording to claim 1, wherein the fatty acid salt is sodium caprylicacid.
 5. The S-nitrosoprotein preparation according to claim 1, whereinthe S-nitrosoprotein is S-nitrosoalbumin.
 6. A method of producing anS-nitrosoprotein, comprising adding a nitrogen monoxide donor to aprotein solution containing a stabilizing agent comprising at least onecompound or a combination of compounds selected from an N-acetylaminoacid, a fatty acid and a fatty acid salt.
 7. The production methodaccording to claim 6, wherein the N-acetyl amino acid isN-acetyltryptophane or N-acetylmethionine.
 8. The production methodaccording to claim 6, wherein the fatty acid is oleic acid or caprylicacid.
 9. The production method according to claim 6, wherein the fattyacid salt is sodium caprylic acid.
 10. The production method accordingto claim 6, wherein the S-nitrosoprotein is S-nitrosoalbumin.
 11. Theproduction method according to claim 6, wherein the nitrogen monoxidedonor is S-nitrosoglutathione.
 12. A pharmaceutical compositioncontaining the S-nitrosoprotein according to claim 1, wherein theS-nitrosoprotein is S-nitrosoalbumin.
 13. The pharmaceutical compositionaccording to claim 12, wherein the pharmaceutical composition isobtained by mixing an albumin solution containing a stabilizing agentcomprising at least one compound or a combination of compounds selectedfrom an N-acetylamino acid, a fatty acid, and a fatty acid salt with anitrogen monoxide donor during a period from 24 hours beforeadministration to just before administration.
 14. A pharmaceuticalcomposition containing the S-nitrosoprotein according to claim 2,wherein the S-nitrosoprotein is S-nitrosoalbumin.
 15. A pharmaceuticalcomposition containing the S-nitrosoprotein according to claim 3,wherein the S-nitrosoprotein is S-nitrosoalbumin.
 16. A pharmaceuticalcomposition containing the S-nitrosoprotein according to claim 4,wherein the S-nitrosoprotein is S-nitrosoalbumin.
 17. A pharmaceuticalcomposition containing the S-nitrosoprotein according to claim 5,wherein the S-nitrosoprotein is S-nitrosoalbumin.